Tuberculosis is the leading causes of death from infectious disease in the world, and the global situation has considerably worsened because of the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) This R01 grant application proposes to develop the immunomodulatory glycolipid alpha-galactosylceramide (alphaGalCer) as a treatment for tuberculosis. Our experiments have shown that mice treated with alphaGalCer following inoculation with Mtb survive longer compared to vehicle treated mice. The main action of alphaGalCer is the activation of immunoregulatory CD1d-restricted iNKT cells, which can in turn stimulate both innate and adaptive immunity. This grant proposal will address fundamental questions about the biology of iNKT cells and investigate ways to take advantage of their immunomodulatory effects following activation by alphaGalCer. By defining the pathways that are activated downstream of iNKT cells, we hope to develop a more rational strategy to exploit the immunomodulatory properties of alphaGalCer. It has potential use in three therapeutic arenas. First, its relatively non-selective immunomodulatory properties and potential to provide protection against a variety of pathogens make it a candidate as a biodefense drug for post-exposure prophylaxis. Second, it could be used in combination with traditional antibiotics in the treatment of infectious disease including multidrug resistant Mtb. Third, it could be used as an adjuvant for vaccines. The murine model is an excellent system to investigate the effect of alphaGalCer on Mtb infection since both CD1d and NKT cells are conserved structurally and functionally between mice and humans. Aim 1 will determine the cellular and molecular pathways activated by alphaGalCer that protect mice from tuberculosis after inoculation with Mtb. Since alphaGalCer has been reported to have adjuvant-like properties, in Aim 2, we will administer alphaGalCer concurrently with anti-mycobacterial subunit or DNA vaccines to determine whether such a combination is synergistic in its ability to induce protective immunity following aerosol challenge with Mtb. Aim 3 will determine whether alphaGalCer can protect mice from an aerosol challenge of Mtb using a post-exposure or treatment model. Finally, in Aim 4 we will derive Mtb-specific CD 1d-restricted T cells from infected mice. This comprehensive program on the study of the role of CD1d and NKT cells in response to tuberculosis includes innovative directions that may have applications for the treatment or prevention of tuberculosis, increase the understanding of NKT cells, pulmonary immunity and have implications for the immunomodulation of other infectious diseases.